Rapid and robust phylotyping of spa t003, a dominant MRSA clone in Luxembourg and other European countries

Abstract

Background: spa typing is a common genotyping tool for methicillin-resistant Staphylococcus aureus (MRSA) in Europe. Given the high prevalence of dominant clones, spa-typing is proving to be limited in its ability to distinguish outbreak isolates from background isolates. New molecular tools need to be employed to improve subtyping of dominant local MRSA strains (e.g., spa type t003).

Methods: Phylogenetically critical, or canonical, SNPs (can-SNPs) were identified as subtyping targets through sequence analysis of 40 MRSA whole genomes from Luxembourg. Real-time PCR assays were designed around target SNPs and validated using a repository of 240 previously sub-typed and epidemiologically characterized Luxembourg MRSA isolates, including 153 community and hospital isolates, 69 isolates from long term care (LTC) facilities, and 21 prospectively analyzed MRSA isolates. Selected isolates were also analyzed by whole genome SNP typing (WGST) for comparison to the SNP assays and other subtyping techniques.

Results: Fourteen real-time PCR assays were developed and validated, including two assays to determine presence of spa t003 or t008. The other twelve assays successfully provided a high degree of resolution within the t003 subtype. WGST analysis of the LTC facility isolates provided greater resolution than other subtyping tools, identifying clusters indicative of ongoing transmission within LTC facilities.

Conclusions: canSNP-based PCR assays are useful for local level MRSA phylotyping, especially in the presence of one or more dominant clones. The assays designed here can be easily adapted for investigating t003 MRSA strains in other regions in Western Europe. WGST provides substantially better resolution than other typing methods.

Figure 1

Whole genome SNP Phylogeny of Assay Development Panel isolates along with fifteen publically available genomes. The corresponding MLST (black) and spa types (red) are noted next to the strains. The phylogenetic relationships are based on a total of 98,768 SNPs out of which 59,284 were parsimony informative with a CI = 0.65. Numbers next to branches are bootstrap values of 100 replicates. Bar length indicates number of SNPs.

Figure 2

Phylogenetic (A) and topology (B) trees of Assay Development strains (spa t003 only). Tree includes strains from initial assay development panel. The phylogenetic relationships are based on a total of 1937 SNPs out of which 527 were parsimony informative with a CI = 0.84. Numbers next to branches are bootstrap values of 100 replicates. Branch locations of first six assay SNP targets are indicated with a red triangle.

Figure 3

Final spa t003 phylogeny with SNP assay targets. The phylogenetic relationships are based on a total of 1154 SNPs out of which 193 were parsimony informative with a CI = 0.98. Branch locations of the 12 validated spa t003 subtyping assays are indicated with a red triangle. Tree includes 29 additional genomes from Assay Validation Panel to increase resolution within t003.

Figure 4

Whole genome SNP typing (WGST) phylogeny of Long Term Care Facility strains with assay results. The phylogenetic relationships are based on a total of 1434 SNPs out of which 686 were parsimony informative. The CI is 0.99. Numbers next to branches are numbers of SNPs. The first number in genome name is the facility ID number and the second number is the isolate ID number. Facility linked clusters are highlighted with brackets. Results of the SNP assays in the inset table indicate strain genotypes.